1. Field of the Invention
Generally, the present invention relates to the detection and measurement of biomarkers in saliva. More particularly, the subject invention describes methods which employ saliva biomarker chemical species from human and animal subjects for detection, quantification, and evaluation of hazardous and toxic chemical or biological agents.
2. Description of the Related Art
The detection and quantification of potentially hazardous or toxic agents and potentially hazardous infectious biological agents are subjects of concern in biomedical and environmental fields as well as to the general public. Humans and animals are exposed to a wide variety of hazardous agents that may or may not be infectious endogenous xenobiotics. For example, on a world wide basis, pesticides have been estimated to cause as many as 500,000 illnesses annually, with as many as 20,000 deaths resulting from pesticide exposure. Information regarding the presence and quantity of hazardous agents within humans or animals, or in the environment where humans or animals may be exposed, is useful in prescribing medical treatments, avoiding further exposure, and assessing levels of environmental contamination.
Several fields of the biomedical sciences are concerned with the characterization and diagnosis of abnormal or pathologic conditions within the body of a human or animal. There are many types of neurological and systemic diseases leading to abnormal levels of specific biochemicals within the body. These biochemicals need to be detected and measured for the purpose of diagnosis. Currently, detection and measurement methods rely on tissue and blood samples which are used to monitor exposure to infectious agents or toxicants in human and animals or the presence of diseases and contaminating, The presently used techniques are generally limited to detecting tissue damage resulting from acute exposure to high levels of an agent, toxin, toxicant, and the like or tissue damage caused by advanced diseases or exposure to chemicals. The sensitivity of procedures utilizing blood or tissue samples is limited by many technical constraints which prevent detection at low levels of chemical agent or at early stages of diseases. For example, the chronic exposure to low levels of some organophosphate compounds and organic solvents can cause delayed neuropathy in human and animals, and the resulting changes in the nervous system cannot be monitored by currently used methods which characterize blood enzymes.
Acetylcholinesterase, carboxylase, and other enzymes present in blood and tissue have been used for many years as biomarkers to monitor the toxicity of agents both in vivo and in vitro. The presence of toxic or reactive agents in blood or tissue samples can often be detected by the degree of inhibition of one or more enzymes that are sensitive to binding and inhibition by those toxic agents.
The present invention introduces the use of human and animal saliva biomarkers for sensitive and non-invasive detection and quantification of chemical and biological agents, usually hazardous substances, including infectious and non-infectious agents. Several advantages are associated with the use of biomarker species that are enzymes from saliva as disclosed herein instead of from blood or tissue as previously carried out. For example, saliva biomarkers are exposed to toxic or reactive agents systemically through the blood and also locally by inhalation and/or ingestion through the mouth, while plasma and tissue enzymes are exposed only by blood uptake. This makes the intensity and the frequency of saliva exposure to toxicants greater than those in blood and tissues.
Another advantage is that saliva contains 0.2-0.5% protein, versus 7.2% in plasma, and the albumen content of saliva is 1% that of plasma. Chemicals or other hazardous agents may undergo nonspecific binding with various plasma proteins due to the high protein concentration, resulting in a reduction in enzyme sensitivity to the effects of the chemicals or other toxic agents. Still another advantage is the relatively small volume of saliva present in humans and animals, resulting in less dilution and higher concentrations of toxic agents. The volume of plasma (5 liters in the adult human male) is much larger than the volume of saliva (0.8-1.0 liter produced per 24 hour period), and the larger blood volume results in higher dilution of the toxic or reactive agents, making them more difficult to characterize.
Yet another advantage provided by oral saliva biomarkers is that the secretory cells of salivary glands are innervated by 5-10 parasympathetic neurons, and saliva probably contains true enzymes such as saliva cholinesterase which differs from the pseudo-cholinesterase present in plasma. Thus, the inhibition of true acetylcholinesterase from saliva is a better indicator for monitoring effects on the nervous system by a chemical agent than inhibition of the equivalent enzyme in plasma.
Additionally, collection of saliva does not require invasive procedures or special equipment as does the collection of tissues, blood, or plasma. The present invention only requires 1-2 mL of saliva for biomarker enzyme analysis.
Methods for protein fractionation and analysis of human saliva and the use of human saliva for detecting pregnancy are known. However, the presence of sensitive biomarkers species such as, but not limited to, acetylcholinesterase, carboxypeptidase, and carboxylase in saliva does not appear to have been reported, and the use of enzyme biomarkers from human and animal saliva for characterization of hazardous or toxic agents has not, it appears, heretofore been disclosed.
Accordingly there is a need for a method of detecting, quantifying, and evaluating hazardous agents which provides for early detection, which can detect low levels of the agents, and which is not invasive and does not require blood or tissue samples from subjects. The present invention satisfies these needs, as well as others, and generally overcomes the deficiencies experienced in the background art.
The present invention pertains to new and sensitive methods which use saliva biomarker species from humans and animals to detect, measure, and quantify the presence of infectious and non-infectious hazardous and reactive agents, and to evaluate the functional status of living tissues. Biomarker species contained in saliva are used in the subject invention to evaluate the toxicity of hazardous agents in two broad applications: (1) in vivo biomedical studies for internal bodily exposure of people or animals to the agents of interest, and (2) in vitro environmental measurements of the presence and/or concentration of the various agents of interest in environmental samples such as water, food and the like. The presence of toxic or reactive agents in samples can be detected, for example, by the degree of inhibition of one or more biomarker enzymes present in saliva. The saliva provides a source of the enzymes that are inhibited by the agents of interest. For the subject invention, the inhibition of biomarker enzymes present in saliva provides a method of evaluating hazardous agents which appears to be more sensitive than currently available assay methods.
Generally, the inventive method comprises the steps of acquiring or otherwise providing a saliva sample, analyzing or measuring the levels or concentrations of one or more biochemical constituents present in the saliva sample, and comparing the levels of the biochemical constituents in the sample with baseline levels or concentrations obtained or determined from control samples, standards, theoretical quantities, or specimens. Biomarker species present in the saliva sample, such as enzymes, antibodies, and other proteins, are responsive to various toxic or hazardous infectious and non-infectious agents, and the measured levels of biochemical constituents indicate the level of response of the biomarkers to the hazardous agents and thus the quantity of hazardous agent present.
In a first or in vivo embodiment of the invention, saliva samples are taken from human and/or animal subjects and biomarkers such as enzymes or antibodies in the saliva are monitored via inhibition or binding. Specifically, the presence of and extent of exposure to hazardous agents is measured by the presence and amount or level of specific chemical or biological constituents, by the degree of enzymes inhibition, by the amount of specific binding, or by the changes in the amount of the biochemical constituents in saliva. Using this embodiment, hazardous agents that inhibit carboxylase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, amylase and other biomarker enzymes present in oral saliva are detected and quantified. Hazardous agents which inhibit these enzymes include, among others, organophosphate and carbamate pesticides and fluorophosphate nerve agents. The amount of hazardous agent present in the saliva is determined by comparison to a standard curve for the hazardous agent which is determined from control samples or theoretical predictions.
In a second or in vitro embodiment, enzymes or other biomarkers from animal or human saliva are used as a ready source for the biomarkers to monitor the presence of toxic or reactive agent in external or environmental samples from tissue, urine, feces, milk, air, water, soil, plants, or other sources. The amount of hazardous agent present in samples is determined from a standard or theoretical inhibition or binding curve for that agent. In one application of this embodiment, acetylcholinesterase and carboxylase (generally defined as enzymes having esterase-like activity) enzymes in human saliva are used to detect the presence of organophosphates such as pesticides in water samples with exceptional sensitivity.
By way of example and not of limitation, the analyzing step preferably is carried out by detecting and monitoring the level or concentration of one or more biochemical constituents in a saliva sample by spectrophotometric or chemical means. The biochemical constituents analyzed are indicative of the response of biomarker enzymes to various hazardous agents. The biochemical constituents may be reactive substrates which are specific for the biomarker enzymes, the reaction products of the biomarker enzymes and their specific substrates, the biomarker enzymes themselves, or other chemical or biological constituents present in a saliva sample which indicate the presence and quantity of a hazardous chemical agent. In the presently preferred embodiments, the analyzing step generally involves detecting inhibition of specific biomarker enzymes by spectrophotometrically monitoring (or monitoring by an equivalent method) reaction product formation and/or depletion of reactive substrates within the samples. The analyzing step also preferably includes measuring the overall amount of protein (this overall amount includes both enzymes and non-enzyme protein components) present in the saliva sample.
The invention may also include the step of determining baseline levels of biochemical constituents from standard or control saliva samples. Generally, standard curves for biochemical constituents which reflect biomarker enzyme activity are prepared from control samples. The determining step also preferably includes determining baseline levels for the overall protein content of saliva samples.
The in vitro embodiment of the invention preferably also includes the steps of obtaining or extracting and concentrating hazardous agents from environmental, tissue, or other samples. Purification and concentration of the saliva sample may also be carried out in this embodiment. Evaluation of the toxicity of new or unknown infectious or non-infectious hazardous agents may be carried out using the in vitro embodiment.
The present invention has scientific value and has several applications with significant commercial potential. The scientific value is exemplified by the discovery of the presence of biomarkers such as acetylcholinesterase and carboxylase enzymes in human saliva and the use of such saliva enzymes to evaluate exposure of human and animals to hazardous agents in both in vivo and in vitro settings. These biomarkers can also be used to monitor changes in human and animal health that result from exposure to endogenous compounds. Purified human and animal saliva enzymes to be used in clinical, research, industrial, and military applications, as well as by the general public to evaluate the, presence, quantity, and toxicity of agents may be provided.
Test kits can be fabricated that utilize the subject methods with related equipment for biomarker enzyme assays. The kits can also be used to detect residue of chemical and biological toxicants in milk and other biological fluids, animal and plant tissues, water, air, and soil samples. Test kits and equipment may be used by agriculture workers, industrial hygienists, military personnel, researchers, and members of the general public.
An object of the invention is to provide a method for detection, quantification, and evaluation of hazardous agents which utilizes biomarkers present in oral saliva of humans and animals.
Another object of the invention is to provide a method for detection, quantification, and evaluation of hazardous agents which is quick and facile to use and which does not require expensive equipment or extensive training.
Another object of the invention is to provide a method for detection, quantification, and evaluation of hazardous agents which allows sensitive and accurate characterization of agents present in low levels.
Another object of the invention is to provide a method for detection, quantification, and evaluation of hazardous agents which allows both in vivo and in vitro characterization of agents.
Another object of the invention is to provide a method for detection, quantification, and evaluation of hazardous agents which is non-invasive and does not require blood or tissue samples.
Another object of the invention is to provide a method for detection, quantification, and evaluation of hazardous agents which may be used in kit forms.
Further objects of the invention will be brought out in the following portions of the specification, wherein the detailed description is for the purpose of fully disclosing the invention without placing limits thereon.